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SAM 107.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 107

Supplemental Assay Method for the Titration of Neutralizing Antibody 

against Selected Bovine Viruses (Constant Serum-Varying Virus Method)

Date: October 16, 2014

Number: SAM 107.04

Supersedes: SAM 107.03, June 28, 2011

Standard Requirement: 9 CFR 113.200

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 24Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the Test Serum Samples

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendices
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

(Constant Serum-Varying Virus Method)

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro serum neutralization (SN) assay

test method to determine the neutralization index (NI) against bovine rhinotracheitis virus (IBR), 

bovine virus diarrhea (BVD) Type 1 and Type 2, parainfluenza (PI3), or bovine respiratory 

syncytial virus (BRSV).

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Incubator, 36°± 2°C, 5% ± 1% CO2, high humidity 

2.1.2 Vortex mixer

2.1.3 Microscope, inverted light

2.1.4 Microscope, fluorescent

2.1.5 Micropipettor, 200-μL, 1000-μL single channel, 5- to 50-μL x 12-channel,

and tips

2.1.6 Water bath

2.1.7 Centrifuge and rotor

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 Indictor Virus

 IBR

 BVD Type 1

 BVD Type 2

 PI3

 BRSV
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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2.2.2 Cell cultures free of extraneous agents as tested by title 9, Code of Federal

Regulations, (9 CFR):

1. Madin-Darby bovine kidney (MDBK) cells used for IBR and PI3 SN 

testing

2. Bovine Turbinate (BT) used for BVD Type 1 and Type 2 testing

3. Embryonic bovine lung (EBL) cells used for BRSV testing

2.2.3 Minimum essential medium (MEM) (National Centers for Animal Health

(NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 2.2 g sodium bicarbonate (NaHCO3)

3. Dissolve Sections 2.2.3(1) and 2.2.3(2) with 900 mL deionized water 

(DI).

4. Add 5 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to 

60° ± 2°C until dissolved. Add to Section 2.2.3(3) with constant stirring.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8-6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add:

a. 10 mL L-glutamine

b. 50 μg/mL gentamicin sulfate

8. Store at 4°± 2°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS)

3. Store at 4°± 2°C.
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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2.2.5 Maintenance Media

1. 980 mL of MEM

2. Aseptically add 20 mL gamma-irradiated FBS.

3. Store at 4°± 2°C.

2.2.6 Monoclonal antibodies (MAb)

1. Anti-BVD Type 1 MAb

2. Anti-BVD Type 2 MAb

2.2.7 Anti-mouse fluorescein isothiocyanate labeled conjugate (Anti-Mouse

Conjugate)

2.2.8 80% Acetone

1. 80 mL acetone

2. 20 mL DI

3. Store at room temperature (23°± 2°C).

2.2.9 0.01 M Phosphate buffered saline (PBS)

1. 1.33 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 2N HCl.

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 4°± 2°C.
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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2.2.10 FBS negative for IBR, BVD, PI3, and BRSV antibodies

2.2.11 Cell culture plate, 96-well

2.2.12 Polystyrene tube, 17 x 100-mm

2.2.13 Serological pipette, 10-mL

2.2.14 Plastic wash bottle, 500-mL

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in antibody titration assays, cell culture maintenance, and in 

the principles of aseptic techniques.

3.2 Preparation of equipment/instrumentation

3.2.1 On day of test initiation, set a water bath at 36°± 2°C.

3.2.2 On day of test initiation, set a water bath at 56°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 Two days prior to test initiation, seed 96-well cell culture plates with 

MDBK cells, in Growth Medium, at a cell count that will produce a confluent 

monolayer after 2 days of incubation at 36°± 2°C in a CO2 incubator. These 

become the MDBK Test Plates.

3.3.2 One day prior to test initiation, seed 96-well cell culture plates with BT 

cells, in Growth Medium, at a cell count that will produce a confluent monolayer 

after 1 day of incubation at 36°± 2°C in a CO2 incubator. These become the BT 

Test Plates.

3.3.3 On the day of the test initiation, seed 96-well cell culture plates with EBL 

cells, in Growth Medium. These become the EBL Test Plates.

3.3.4 On the day of test initiation, rapidly thaw a vial of an appropriate Indicator 

Virus in a 36°± 2°C water bath. The number of dilutions depends upon the 
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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predetermined titer of the Indicator Virus. Prepare serial tenfold dilutions as 

follows:

1. Pipette 9.0 mL of MEM with a serological pipette into 8, 17 x 100-mm 

polystyrene tubes labeled 10-1to 10-8.

2. Transfer 1.0 mL of an Indicator Virus to the 10-1tube; mix by 

vortexing. Discard the pipette.

3. Transfer 1.0 mL from the 10-1tube to the 10-2tube; mix by vortexing. 

Discard the pipette.

4. Repeat Section 3.3.4(3) for each of the subsequent dilutions, 

transferring 1.0 mL from the previous dilution to the next dilution.

3.3.5 On the day of BT Test Plate examination, dilute Anti-BVD MAb and 

Anti-Mouse Conjugate in PBS according to the manufacturer’s recommendations.

3.4 Preparation of the Test Serum Samples 

On day of test initiation, heat-inactivate all Test Sera in a 56°± 2°C water bath for 

30 ± 5 minutes.

4. Performance of the Test

4.1 Add 150 μL/well of an undiluted Test Serum into a column of a 96-well cell 

culture plate, which becomes the Dilution Plate (Appendix I).

4.2 Add 150 μL/well of FBS into a column of a Dilution Plate.

4.3 Add 150 μL/well of the last 4 dilutions of the Indicator Virus to a row of the 

Dilution Plate.

4.4 Mix by tapping the edge of the Dilution Plate with fingers. Incubate for 60 ± 10 

minutes at room temperature to allow for neutralization of the Indicator Virus.

4.5 At the end of the incubation period, decant Growth Medium from the MDBK Test 

Plates. 

Note: The Growth Media is not removed from the BT and EBL Test Plates.

4.6 Inoculate 50 μL/well of each Virus-serum mixture into 5 wells/dilution of the 

appropriate Test Plate (Appendix II). 
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Note: MDBK Test Plates are used to determine the SN titer against IBR and PI3. BT 

Test Plates are used for BVD. EBL Test Plates are used for BRSV.

4.7 Maintain 5 or more wells on each Test Plate as uninoculated cell controls.

4.8 Inoculate 25 μL/well of the last 4 dilutions of the appropriate Indicator Virus to 5 

wells/dilution as an endpoint titration.

4.9 Incubate MDBK Test Plates for 60 ± 10 minutes at 36°± 2°C in a CO2 incubator.

4.10 Add 200 μL/well of Maintenance Medium to all wells (do not remove virusserum inoculum) of the MDBK Test Plate.

4.11 Incubate the MDBK and BT Test Plates for 4 days ± 12 hours postinoculation at 

36°± 2°C in a CO2 incubator.

4.12 Incubate the EBL Test Plate for 6 days ± 12 hours postinoculation at 36°± 2°C in 

a CO2 incubator.

4.13 At the end of the incubation period, examine the wells with an inverted light 

microscope. Record the number of wells/dilution showing any characteristic CPE for 

IBR, PI3, or BRSV for Test Serum, FBS, and Indicator Viruses.

4.14 An indirect fluorescent antibody technique (IFA) is conducted to determine the 

SN titer against BVD as follows:

4.14.1 Decant media from the BT Test Plates.

4.14.2 Fill wells with 80% Acetone.

4.14.3 Incubate at room temperature for 15 ± 5 minutes.

4.14.4 Decant the 80% Acetone from the BT Test Plate; air dry at room 

temperature.

4.14.5 Pipette 50 μL/well of a diluted Anti-BVD MAb into a BT Test Plate; 

incubate for 45 ± 15 minutes at room temperature.

4.14.6 Rinse by filling the wells completely with PBS; decant the liquid.

4.14.7 Repeat Section 4.14.6 for a total of 2 washes.
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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4.14.8 Pipette 50 μL/well of the diluted Anti-mouse Conjugate into the BT Test 

Plates; incubate for 45 ± 15 minutes at room temperature.

4.14.9 Repeat Section 4.14.6 for a total of 2 washes.

4.14.10 Dip the plate in DI, decant; allow to air dry or dry at 36°± 2°C.

4.14.11 Examine wells with a fluorescent microscope.

4.14.12 A well is considered positive if typical cytoplasmic, apple-green 

fluorescence is observed.

4.14.13 Record the number of wells/dilution showing any characteristic 

fluorescence for the Test Serum, FBS, and Indicator Virus.

4.15 Calculate the TCID50 of the Test Sera, FBS, and Indicator Virus using the 

Spearman-Kärber method as commonly modified.

4.16 The neutralizing ability is determined by subtracting the log of the titer obtained 

with the Test serum from the titer obtained with the Indicator Virus.

Example: Log10 TCID50 Indicator Virus titer 6.0

Log10 TCID50 titer with Test Serum - 2.7

Neutralizing ability = 3.3 logs

5. Interpretation of the Test Results

For a valid test:

5.1 No visible contamination or serum toxicity should be observed in any well or any 

dilution of a Test Sera or the FBS.

5.2 The titer of the FBS should be negative for neutralizing antibody against IBR, 

BVD, PI3, or BRSV.

5.3 The endpoint titration of the Indicator Virus should fall within 2 standard 

deviations of its mean titer as determined by a minimum of 10 previous titrations.

6. Report of Test Results

Record the neutralizing ability titers for the Test Serum on the test record.
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Supplemental Assay Method for the Titration of Neutralizing Antibody against Selected Bovine Viruses 

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7. References

7.1 Title 9, Code of Federal Regulations, parts 113.215 and 113.216, U.S. 

Government Printing Office, Washington, DC.

7.2 Finney DJ. Statistical Method in Biological Assay. 3

rd ed. 1978. Charles Griffin 

and Co., London.

7.3 Rose NR, H Friedman, JL Fahey, eds. Manual of Clinical Laboratory 

Immunology. Chapter 11: Neutralization Assays. 1986. ASM, Washington, D.C.

8. Summary of Revisions

Version .04

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .03

 The Contact information has been updated.

 References to NVSL have been changed to NCAH throughout the document.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes impacting the test were 

made from the previous protocol, the following changes were made to the document: 

 The Contact has been changed from Larry Ludemann to Joseph Hermann.

 The testing format has been changed from roller tubes and Leighton tubes to a 96-

well plate format.

 The terminology of identifying types of BVD has been changed from BVD Type I to 

BVD Type 1 and from BVD Type II to BVD Type 2, to conform to a common 

format.

 2.2: Bovine Turbinate cells have been substituted for embryonic bovine kidney cells 

for BVD Type 1 and Type 2 testing to conform to cells commonly used for BVD 

testing.
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 2.2.3: The description of antibiotics added to the Diluent Medium has been changed 

to reflect the antibiotics used. Penicillin and streptomycin have been replaced by 

gentamicin sulfate, and Amphotericin B has been eliminated from the formulation.

 4.13: The provision for conducting a hemadsorption test for detecting antibody 

against PI3 has been replaced by detecting antibody by CPE.

 4.14: The fluorescent antibody test for detecting antibody against BVD has been 

replaced with an IFA to allow the differentiation between Type 1 and Type 2 BVD 

antibodies. Indicator Virus has been added for clarification purposes.

 4.15: Indicator Virus has been added for clarification purposes.

 4.16: “The neutralizing ability is determined by subtracting the log of the titer 

obtained with the Test serum from the titer obtained with the FBS” has been changed 

to “The neutralizing ability is determined by subtracting the log of the titer obtained 

with the Test serum from the titer obtained with the Indicator Virus”.
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Appendix I

Dilution Plate

 1 2 3 4 5 6 7 8 9 10 11 12

A IV

10-3

TS1 TS2 TS3 TS4 TS5 TS6 TS7 TS8 TS9 TS10 TS11 FBS

B IV 

10-4

C IV

10-5

D IV 

10-6

E IV 

10-3

TS12 TS13 TS14 TS15 TS16 TS17 TS18 TS19 TS20 TS21 TS22 TS23

F IV 

10-4

G IV 

10-5

H IV 

10-6

IV = Indicator Virus dilution, TS = Test Serum, NC = FBS

Appendix II 

Test Plate

1 2 3 4 5 6 7 8 9 10 11 12

A 

10-3

TS1 TS1 TS1 TS1 TS1 CC TS2 TS2 TS2 TS2 TS2 CC

B 

10-4

C 

10-5

D 

10-6

E 

10-3

TS3 TS3 TS3 TS3 TS3 CC TS4 TS4 TS4 TS4 TS4 CC

F 

10-4

G 

10-5

H 

10-6

TS= Test Serum, CC= Cell Control